Methods for producing polypeptides by host organisms transformed with recombinant DNA molecules are well known (for a general review, see the Feb. 11, 1983 issue of Science, 219:535 entitled "Biotechnology"). Such recombinant production methods generally comprise preparing a DNA sequence encoding a polypeptide of interest, inserting it into an expression vehicle (e.g. a virus or a plasmid) to produce a recombinant DNA molecule in which the DNA sequence is under the control of an expression control sequence, transforming a suitable host organism with the recombinant DNA molecule, and culturing the host to produce the polypeptide of interest.
The level of recombinant production of polypeptides in a host cell is governed by four major factors: (1) the number of copies of the DNA sequence encoding the polypeptide, (2) the efficiency with which those DNA sequences are transcribed to messenger RNA ("mRNA"), (3) the efficiency with which the mRNA is translated to polypeptides, and (4) the stability of the polypeptides in the host cell. Transcription and translation together comprise expression of a DNA sequence. Efficiency of transcription in turn is dependent upon, inter alia, various sequences such as, for example, promoters and effector binding sites that serve as loci to initiate mRNA synthesis. Transcription terminator sequences ("transcription terminators") are stop sites for RNA polymerase which halt or regulate transcription. Transcriptional regulation is achieved by modulating the efficiency with which the RNA polymerase can recognize and interact with a given DNA sequence to initiate or to terminate RNA transcription (Rosenberg and Court, 1979, Ann. Rev. Genet. 13:319). For example, such interaction of promoter and terminator regions may be observed in the transcriptional regions of some E. coli plasmids where transcription terminators appear to balance transcription initiated by promoters of different strengths (Stuber and Bujard, 1981, Proc. Natl. Acad. Sci. USA 78:167).
This interaction of promotion and transcription termination has also been observed in the cloning of very strong promoters. For example, although strong promoters are desirable for increasing the expression level of a DNA sequence, such promoters may interfere with replication of plasmids containing them. Stable plasmids containing strong promoters from bacteriophage T5 have been constructed using natural transcription terminators downstream from the strong promoters. In the absence of such terminators, it had not been possible to clone these strong T5 promoters (Gentz et al., 1981, Proc. Natl. Acad. Sci. USA 78:4936). Finally, transcription termination may be useful in avoiding the potential disadvantages of the expression or control of a particular gene sequence by "read-through" transcription from promoters in other regions of the expression vector.
Several natural transcription terminators are known. They are divided generally into two classes, independent, and factor-dependent transcription terminators (Holmes et al., 1983, Cell 32:1029. Independent transcription terminators may function independently of other products as stop sites for RNA polymerase. In vivo independent transcription terminators are usually located at the ends, within and between the genes of operons. For example, independent transcription terminators are found near the beginning of biosynthetic operons that are regulated by attenuation, such as the trp operon (Holmes et al., supra). As attenuators, independent terminators regulate expression of downstream cistrons. Independent terminators have also been found to control other types of transcriptional units. Although most independent transcription terminators function only in a single orientation, some bidirectional transcription terminators are also known to exist (Holmes et al., supra).
Generally, independent transcription terminator regions are structurally similar. For example, most terminators include a GC-rich sequence preceding the termination site and a sequence of T-residues in the nontemplate DNA strand attached to the termination site. The RNA polymerase traverses the GC-rich sequence to produce mRNA which can form a stable base-paired stem-and-loop structure within the mRNA. Transcription then usually terminates just downstream from the stem-and-loop structure where the T-residues result in a RNA ending with a sequence primarily comprising uridylate residues (Rosenberg and Court, supra; Holmes et al., supra; and Brennan and Geiduschek, 1983, Nucl. Acids Res. 11:4157).
Although these regions of independent transcription termination have certain similarities, it is extremely difficult, if not impossible, to predict from DNA sequence information alone which regions of DNA will be recognized by RNA polymerase as a transcription terminator. Neither is it yet understood how nucleotide similarities observed at the primary sequence level contribute to specifying a transcription termination function (Rosenberg and Court, supra).
The second general class of transcription terminators is the factor-dependent terminators. Factor-dependent terminators require ancillary products or factors to function as stop sites for RNA polymerase. Factor-dependent terminators comprise a large and diverse group of DNA sequences that are very complex in structure and function (Holmes et al., supra).